Synthesis of homopteroic and homofolic acid

ABSTRACT

THE PRODUCTION OF HOMOPTEROIC AND HOMOFOLIC ACIDS BY VIRTUE OF SELECTING A CRITICAL KETO CONDENSING AGENT, NAMELY 1 - ACETOXY-4-(N-ACETY) - (P - CARBETHOXYPHENYL) AMINO)-2BUTANONE, WHICH IS CONDESED WITH A CONVENTIONAL PYRIMIDINE REACTANT, NAMELY 6-HYDROXY-2,4,5-TRIAMINOPYRIMIDINE, UNDER CONDITION OF NARROW PH CONTROL, PREFERABLY IN THE MILDLY ALKALINE RANGE 9.0-9.4.

United States Patent 3,637,695 SYNTHESIS OF HOMOPTEROIC AND HOMOFOLIC ACID Young-Ho Kim, Vytautas Grubliauskas, and Orrie M. Friedman, Waltham, Mass, assignors to the United States of America as represented by the Secretary of Health, Education, and Welfare No Drawing. Filed Mar. 20, 1968, Ser. No. 714,426 Int. Cl. C07d 57/28 US. Cl. 260-2515 2 Claims ABSTRACT OF THE DISCLOSURE The production of homopteroic and homofolic acids by virtue of selecting a critical keto condensing agent, namely 1 acetoxy-4-[N-acetyl (p carbethoxyphenyl) aminoJ-Z-butanone, which is condensed with a conventional pyrimidine reactant, namely 6-hydroxy-2,4,5-triaminopyrimidine, under condition of narrow pH control, preferably in the mildly alkaline range 9.0-9.4.

Homofolic and homopteroic acids are homologs of folic and pteroic acids containing an additional methylene group between the pteridine and p-aminobenzoic acid moieties or fractions thereof. Thus, they may be considered as analogs or in some teachings as next upward homologs of the older compounds. The present process is designed to more effectively produce these compounds which are of greater current interest. Homofolic acid is a potent inhibitor of specific bacteria and tumors, and homopteroic acid has been found to be effective against Plasmodium cynomalgi and a pyrimethamine-resistant variant of this organism. Dr. Leon Goodman (Goodman process, supra) and his associates devised an elegant method involving more than 18 steps for the production of homofolic acid but reported yields by this synthesis were small and several of the steps are not suitable for large scale production. Additionally, the present process eliminates several steps of Goodman and is commercially feasible.

Patent art involving keto condensing agents and related art includes: 2,443,165, Hultquist et al.; 2,472,481, Hultquist et al.; 2,558,711, Weisblat et al.; 2,674,617, Weisblat et al.; 2,760,960, Gazzola et al.

The difficulty with the prior art compounds using keto reactants has been that the unwanted 7-position isomer of the pteridine fraction was produced concurrently with the 6-position isomer and separation was difiicult. This is remedied in the present invention by the selection of a critical coupling agent plus utilization of a carefully controlled mildly alkaline medium (pH 9.0-9.4), therewith directing the condensation to the 6-substituted pteridine. In the first stage of the process, the invention proceeds by a short-cut method of condensing the conventional and known 6-hydroxy-2,4,S-triaminopyrimidine with the novel and critical 1 acetoxy-4- [N-acetyl-(p-carbethoxyphenyl) amino]-2-butanone in approximately equi-molar amounts. This step of the process is carried out preferably under inert conditions, e.g., a nitrogen blanket, although neon, argon, etc., may also be used. The preferred time and temperature limitations were about 12-16 hours (narrow) and -40 hours (broad) at a preferred 60 C. or a broad 60- l0 C. In this novel step the dihydroester is produced which is oxidized and saponified as by hydrogen peroxide and a base to give the overall yield of the homopteroic acid of 40-50% (average), substantially above that produced by the prior art and free of unwanted isomer. The production of the homopteroic acid from the blocked ester may also be accomplished by alternate methods readily understood by the chemist skilled in the art.

The conversion of homopteroic to homofolic acid is ac complished by following the procedure of Goodman, supra,

Patented Jan. 25, 1972 in J. Org. Chem., 30, 3404 (1965), in which sensitive N position is blocked by utilization of -COCF as a substituent by means of trifluoroacetic anhydride. Again as in the homopteroic acid step, the free acid must be isolated after saponifying by removal of the substituent from the N N and the ester substituents from the terminal carboxy groups of the glutamic fraction. Any convenient hydrolysis procedure involving a mild base may be used and this conventional procedure is believed to be within the competence of a chemist skilled in the art.

EXAMPLE 1 Preparation of 1-acetoxy-4-[N-acetyl- (pcarbethoxyphenyl) amino1-2-butanone A solution of 140.2 g. of 4-[N-acety-(p-carbethoxyphenyl) amino]-1-bromo-2-butanone in 780 ml. of absolute ethanol was added dropwise to 156 g. of tetraethylammonium acetate in 466 ml. of absolute ethanol during a 30-minute period, with stirring, at room temperature. The reaction mixture was stirred for an additional 30 minutes at room temperature and the solvent was removed in vacuo at 30-35 C.

The yellowish residue was suspended in 2 liters of water and the water-insoluble material was extracted with five 550 ml. portions of methylenechloride. The combined methylene chloride extracts were stirred with 660 g. of Florisil (Floridin Company product), 26.4 g. of charcoal (Norit A, neutral and Nuchar in a 1:1 ratio), and 50 g. of anhydrous sodium sulfate for 1 hour. The mixture was filtered on a Biichner, was washed thoroughly with methyl ene chloride and was freed from residual charcoal by gravity filtration.

The solvent was removed from the filtrate by evaporation under reduced pressure to give 106.3 g. (80.7%; the best yield was of a light yellow oily product, 21 1.5225.

Analysis.Calcd. for C H NO (percent): C, 60.8; H, 6.3; N, 4.2. Found (percent): C, 60.8; H, 6.4; N, 4.1.

EXAMPLE 2 Preparation of diethyl N -acetyl-5,6- dihydrohomopteroate A suspension of 42.9 g. of 6-hydroxy-2,4,5-triaminopyrimidine sulfate in 1 liter of water containing 43.8 g. of barium chloride was stirred at for 30 min. After the mixture was cooled to room temperature the precipitated barium sulfate was removed by filtration and thoroughly washed with 500 ml. of water. The triaminopyrimidine hydrochloride thus obtained was added to 1.5 liters of 4 M sodium acetate containing 31.5 g. of cysteine hydrochloride. To the above mixture was added a solution of 66 g. of 1-acetoxy-4[N-acetyl(p-carbethoxyphenyl) amino]-2-butanone in 2 liters of p-dioxane and the solution was adjusted to pH 9.0 with triethylamine.

The resultant mixture was heated at 60, with stirring, under a nitrogen atmosphere for 14 hours. The solution was concentrated to about 3 liters by evaporation in vacuo and was acidifide to pH 7 with 2 N hydrochloric acid. Refrigeration of the mixture for 72 hours gave a tancolored solid which was collected by filtration and washed with water and ether to give 29.1 g. of the desired product. An additional 2.8 g. of the product was collected by concentration of the mother liquor to about 2 liters at pH 9.0 followed by neutralization to pH 7, and then refrigeration overnight. Total yield was 31.9 g.

(44.8%) M3,? 253, 278 (shoulder), and my EXAMPLE 3 Diethyl N -acetylhomopteroate A suspension of 5.31 g. of diethyl N -acety1-5,6-dihydrohomopteroate in 139 ml. of 0.4 N-hydrochloric acid was stirred at room temperature for 30 min. The turbid solution thus obtained was filtered and a solution of 2.6 ml. of 30% hydrogen peroxide in 11.5 ml. of water was added dropwise to the clarified filtrate during a one-min. period. The mixture was stirred at room temperature for 90 min. and was neutralized to pH 7 with 6 N-ammonium hydroxide. The precipitate thus obtained was collected by filtration and washed with 20 ml. of water, then with 10 ml. of cold ethanol, and finally was dried in vacuo to afford 4.75 g. (90%) of a yellow solid product.

1335 253 and 366 my.

EXAMPLE 4 Homopteroic acid A solution of 7.66 g. of diethyl N -acetylhomopteroate in 116.5 ml. of 10% sodium hydroxide was heated on steam bath under nitrogen atmosphere for 2.5 hours. The resultant red-brownish solution was chilled at 10 for 3 hours and the precipitated sodium salt was collected by centrifugation. The yellow solid was dissolved in 50 ml. of hot water and the solution was adjusted to pH 3.2 with 1 N-hydrochloric acid. The resulting gelatinous mass was diluted to a volume of 200 ml. with water, then stirred at C. for 1 hour. The product was collected by centrifugation, then washed twice by centrifuging with water. The crude product thus obtained was washed again with ethanol-ether (2: 1) and collected by filtration to give 5.57 g. (87%) of a yellow solid my 255, 277 and 365 m Analysis.Calcd. for C H N O (percent): C, 55.2; H, 4.3; N, 25.8; ash, 0.0. Found (percent): C, 54.7; H, 4.3; N, 25.8; ash, 0.0.

EXAMPLE 5 The condensation reaction between 6-hydroxy-2,4,5-triaminopyrimidine and l-acetoxy 4 [N-acetyl-(p-carbethoxyphenyl)amino]-2-butanone was carried out using the following procedure: a solution of 6-hydroxy-2,4,5-triaminopyrimidine hydrochloride was added to a solution of cysteine hydrochloride in 4 M sodium acetate solution. The mixture thus obtained was treated with a solution of 1-acetoxy-4- [N-acetyl- (p-carbethoxyphenyl) amino] -2 butanone in dioxane and adjusted to pH 9.2 with triethylamine. After stirring under nitrogen for 14 hours at 60 C., the solution was concentrated by evaporation under reduced pressure, neutralized to pH 7 with hydrochloric acid, and refrigerated to yield the dihydrohomopteroate as a yellow powder. The free homopteroic acid was obtained by oxidizing with hydrogen peroxide together with subsequent Saponification and an overall yield of different runs was from 40-50% of homopteroic acid. The following comparison illustrates graphically the positive identification of the product as compared with the samples produced by the method of Goodman, supra.

UV SPECTRA OF HOMOP'IEROIC ACID Authentic sam- 4 EXAMPLE 6 Homofolic acid By following the procedure of Goodman, supra, the homopteroic acid of Example 5 was converted to homefolic acid; after blocking N with the trifiuoroacetyl group followed by blocking of N with the acetyl group, it was treated with diethyl L-glutamate in the presence of isobutyl chloroformate. Saponification of the resultant product with 0.2 N sodium hydroxide gave the free acid.

Analysis.Calcd. for C H NqOg' /2HO (percent): C, 51.7; H, 4.7; N, 21.1; ash, 0.0. Found (percent): C, 51.3; H, 4.7; N, 20.7; ash, 0.0.

As in Example 5, comparison with a known sample, produced by the Goodman et al. procedure, supra, showed the following results:

UV SPECTRA OF HOMOFOLIC ACID Authentic sample prepared by the method pH of Goodman Present M... e) et a1. E invention (6) PAPER CHROMATO GRAPHY Authentic sample prepared by the method of Goodman Present et al. invention Method: Ascending. Paper: Whatman #1. Solvent: 0.1 M

The homofolic acid produced by the process of the present invention, when tested for biological activities, gave activities consistent with the known utilities of homofolic acid. When reduced to the dihydro variety it inhibited Escherichia coli thymidylate synthetase and when further reduced catalytically to the tetrahydro variety, it inhibited the growth of Streptococcus faecalis.

A summary of the biological data of homofolic acid produced by the present process is illustrated as follows:

BIOLOGICAL DATA OF HOMOFOLIC ACID Inhibition of E. Inhibition of 0011', thymidyl- Signal spectral S. jaecalis ate synthetase peak at 295 m Sample 7 /ml. for 50 a (M for percent in UV (pH 7 in Lot No inhibition inhibition) 2M-IISCI'I2CHOII) 0. 2 2. 2X10- l 0.27 2. 3 l0 l 0. 25 2. 0X10- l Authentic sample J. Am. Chem. 500., 86, 308 (1964).

UNITED STATES PATENTS 2,472,481 6/ 1949 Hultquist et al 260251 ALEX MAZEL, Primary Examiner R. V. RUSH, Assistant Examiner 

